PROJECT SUMMARY/ABSTRACT The menopausal hormone therapy (HT) timing hypothesis was recently validated in a newly completed NIA- funded randomized controlled trial, the Early Versus Late Intervention Trial with Estradiol (ELITE) that showed that HT administered <6 years-since-menopause significantly reduced subclinical atherosclerosis progression relative to placebo, whereas there was no effect on progression in women who received HT >10 years-since- menopause. Thus, while the literature supports and ELITE validates HT as a potential treatment-specific and age-related opportunity for reducing cardiovascular disease and all-cause mortality trends in women, the biological mechanisms underlying the age-related atheroprotective effects of HT when administered early versus late after menopause are not known. This proposal seeks to address these fundamental gaps in knowledge by leveraging the design and rich dataset of ELITE to investigate the clinical biomarkers and molecular mechanisms of carotid artery intima-media thickness (CIMT) progression as a function of the timing of HT initiation relative to menopause. Based on our prior studies and evidence from the literature, our overall hypothesis is that HT initiation <6 years-since-menopause has favorable effects on the bioavailability and signaling of sex hormones and atherosclerosis-related inflammatory biomarkers in the circulation, which leads to reduced CIMT progression. We also hypothesize that the molecular mechanisms for the divergent atherosclerosis outcomes in ELITE can be identified through longitudinal analyses of mRNA gene expression and DNA methylation status of selected candidate genes in blood cells, which can vary as a function of age- related processes. In Aim 1, we will determine whether biomarkers of sex hormone bioavailability and inflammatory pathways potentially regulated by HT can explain the differential effect of HT on subclinical atherosclerosis progression according to time-since-menopause. Using clinical data that already exists and that will be developed from ELITE participants using stored samples, we will determine the longitudinal relationship between blood levels of sex hormone binding globulin, sex hormones and atherosclerosis-related inflammatory biomarkers measured at baseline, 6, 12, 24 and 48 months with CIMT progression as a function of early versus late HT intervention. In Aim 2, we will determine whether longitudinal changes in mRNA expression levels and methylation status of genes encoding estrogen receptors and a panel of inflammatory molecules in blood cells are explanatory molecular mechanisms for the modification of atherosclerosis progression by time-since-menopause when HT is initiated. Understanding mechanism(s) of this sex-specific and age-related opportunity for reducing CVD and all-cause mortality is key to optimizing HT and instrumental for new drug discovery. The implications of estrogen deficiency on the rates of CVD are of enormous public health importance. As such, this proposal has high clinical and